Mitotracker dye

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

Mitotracker dye is a fluorescent probe used to stain and visualize mitochondria in live cells. It accumulates in active mitochondria, allowing for the detection and analysis of mitochondrial structure and function.

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MitoTracker (469 citations) MitoTracker Red CMXRos dye (30 citations) MitoTracker Green dye (27 citations) MitoTracker Green FM dye (21 citations) MitoTracker Red dye (15 citations) MitoTracker Red FM Dye (8 citations) MitoTracker Deep Red dye (8 citations) Mitotracker Orange dye (4 citations) MitoTracker Deep Red FM dye (2 citations) MitoTracker green fluorescent dye (2 citations)

36 protocols using mitotracker dye

Immunofluorescence Imaging of Mitochondrial Morphology

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Cells were fixed in 4% paraformaldehyde in PBS for 15’ at RT, washed and permeabilized in 0.25% Triton-X-100 in PBS for 5’. Blocking was performed in 5% goat serum in PBS for 1 hour. Incubation with primary antibodies was at 4̊ C for 16h. Primary antibodies used was MHyC (Sigma, MY32, 1:500) and secondary antibody AlexaFluor 568 conjugated anti-mouse (Invitrogen, 1:1000). Nuclei were stained by DAPI (Sigma, 1µg/mL).
For the detection of mitochondrial shape Scr, sh1-p63 and sh2-p63 cells were incubated with Mitotracker dye (50nM, Invitrogen) together with Hoesch 33342 (1μM, Sigma-Aldrich) for nuclei staining, for 30 minutes at 37̊C.
Cell images were obtained by confocal laser microscope NIKON Eclipse Ti using EZ C.1 software (Nikon).

Ciuffoli V., Lena A.M., Gambacurta A., Melino G, & Candi E. (2018). Myoblasts rely on TAp63 to control basal mitochondria respiration. Aging (Albany NY), 10(11), 3558-3573.

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Immunofluorescence Analysis of Germ Cells

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For an IFA, human germ cells were treated with 0.1% Triton X-100, washed twice with Tris-buffered saline (TBS), and subsequently incubated with a primary antibody (PRM2: Santa Cruz, sc-23104; NDC1: Santa Cruz, sc-161929; PCM1: Santa Cruz, sc-50164; OBSL1: Santa Cruz, sc-241578; SPAG4: Santa Cruz, sc-85927; SEPT12: Abnova, H00124404-B01P; LAMINB1: abcam, ab16048) for 60 min at room temperature. After being washed with TBS, the sections were exposed to the Alexa Fluor 568 donkey antigoat IgG antibody (Invitrogen, cat no. A-11057, USA), Alexa Fluor 488 donkey antimouse IgG antibody (Invitrogen, cat no. A-21202, USA), or Fluor 568 donkey antirabbit IgG antibody (Invitrogen, cat no. A-10042, USA) for 60 min at room temperature and washed again with TBS. A MitoTracker dye (Invitrogen, M7514, USA) and 4',6-diamidino–2-phenylindole (Invitrogen, D3571, USA) were used to stain the mitochondria and nuclei, respectively. The midpieces of the spermatozoa were stained using the mitochondrial tracker (Invitrogen, M7514, USA), and 4',6-diamidino–2-phenylindole (DAPI; Invitrogen, D3571, USA) was used to stain the nuclei. Labeled spermatozoa were examined and images were captured using the upright microscopy system BX60 (Olympus, Tokyo, Japan), and MetaMorph image analysis software was used to analyze the acquired images.

Yeh C.H., Kuo P.L., Wang Y.Y., Wu Y.Y., Chen M.F., Lin D.Y., Lai T.H., Chiang H.S, & Lin Y.H. (2015). SEPT12/SPAG4/LAMINB1 Complexes Are Required for Maintaining the Integrity of the Nuclear Envelope in Postmeiotic Male Germ Cells. PLoS ONE, 10(3), e0120722.

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Cell Line Maintenance and Reagents

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HEK293T, HEK293 cells stably expressing hTLR2 (HEK-TLR2) and HEK293 cells stably expressing MD2, CD14 and hTLR4 (HEK-TLR4) were purchased from Invivogen and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and appropriate antibiotics at 37°C, 5% CO₂, under humidified environment. Antibodies to GAPDH and β tubulin were from Santa Cruz Biotechnology, LC3B was from Cell Signaling Technology, His and FLAG were from Sigma-Aldrich, rabbit IgG Alexa Fluor 568 conjugate was from Invitrogen. E. coli 055:B5 lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were from Sigma-Aldrich. Mitotracker dye was from Invitrogen.

Carlsson E., Thwaite J.E., Jenner D.C., Spear A.M., Flick-Smith H., Atkins H.S., Byrne B, & Ding J.L. (2016). Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy. PLoS ONE, 11(7), e0158575.

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Cell Viability Evaluation via AO/EB and MitoTracker

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The viability of cells in chambers 1 and 3 was evaluated using an acridine orange/ethidium bromide (AO/EB) double fluorescence kit (Solarbio). The cells in chambers 2 and 4 were incubated in AO/EB solution (1:1) for 2–5 min, and the number of viable cells was counted under an inverted fluorescence microscope (Olympus). In addition, the cells were incubated in MitoTracker dye (Invitrogen, Carlsbad, CA, USA) for 15 min, and the viability of cells was determined based on the mitochondrial mass.

Zhao Y., Wang D., Xu T., Liu P., Cao Y., Wang Y., Yang X., Xu X., Wang X, & Niu H. (2015). Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment. Oncotarget, 6(36), 39196-39210.

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Mitochondrial Superoxide Imaging in Brain

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Mito-SOX red fluorescent dye was employed to examine in vivo mitochondrial O₂^•− radical formation in whole brain tissues. Mito-SOX fluorescent dye is a live cell-permeable dye that specifically targets mitochondria and shows red fluorescence after being oxidized via O₂^•− radicals. In brief, whole brains (~10 nos.) were dissected in 1XPBS and thereafter stained with Mito-SOX dye (5.0 µM) (Catalogue no.: M36008; Invitrogen, Carlsbad, CA, USA) and Mitotracker dye (100.0 nM) (Catalogue no.: M7514; Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature. After washing three times with 1XPBS for 5 min, tissue samples were mounted in Vectashield mounting media with DAPI (Vector Laboratories, Germany), and immediately images of samples were captured by using a confocal laser scanning fluorescence microscope (Leica TCS-SPE Microsystems, Wetzlar, Germany) with 63X objective.

Chaouhan H.S., Li X., Sun K.T., Wang I.K., Yu T.M., Yu S.H., Chen K.B., Lin W.Y, & Li C.Y. (2022). Calycosin Alleviates Paraquat-Induced Neurodegeneration by Improving Mitochondrial Functions and Regulating Autophagy in a Drosophila Model of Parkinson’s Disease. Antioxidants, 11(2), 222.

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Immunostaining of Mitochondrial Proteins

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In brief, cells were fixed in 4% PFA (Sigma-Aldrich) for 15 min, followed by a 10-min permeabilization step (0.1% Triton X-100 in PBS for internal cell markers). The blocking step was performed by incubation in 2% BSA for 30 min. Cells were incubated with primary antibodies at 4°C overnight and further incubated with secondary antibodies for 1 h. For MitoTracker dye staining, cells were cultured in the normal media with 150 nM MitoTracker red (Invitrogen) for 30 min before further fixation and immunostaining. Antibodies against the following proteins were used at the indicated dilutions: CHCHD2 (HPA027407, 1:200; Sigma-Aldrich), SOX1 (4194S, 1:200; Cell Signaling Technology), NESTIN (MAB5326, 1:200; EMD Millipore), TUJ1 (MRB-435P, 1:500; Covance), mtTFA (ab119684, 1:500; Abcam), anti–mouse IgG-FITC (1:800; Sigma-Aldrich), and anti–rabbit IgG-Cy3 (1:1,000; Jackson ImmunoResearch Laboratories, Inc.). Nuclei were labeled with DAPI (Thermo Fisher Scientific). Colocalization coefficient studies were performed using ImageJ software by calculating Manders’ colocalization coefficient, which describes the amount of colocalizing pixels of GFP using pixels generated by RFP.

Zhu L., Gomez-Duran A., Saretzki G., Jin S., Tilgner K., Melguizo-Sanchis D., Anyfantis G., Al-Aama J., Vallier L., Chinnery P., Lako M, & Armstrong L. (2016). The mitochondrial protein CHCHD2 primes the differentiation potential of human induced pluripotent stem cells to neuroectodermal lineages. The Journal of Cell Biology, 215(2), 187-202.

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Visualizing GFP-labeled fungal hyphae

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Conidia (10³) of the respective GFP-labeled strains were cultured in 35-mm cover-glass-bottom petri dishes containing 3 ml of liquid GMM and incubated for 16 h at 37°C prior to visualization. For mitochondrial staining, where noted, 3 μl of MitoTracker dye (Invitrogen) was added to the growth medium 15 min prior to visualization. Hyphae were visualized using an Axio Observer 3 microscope (Carl Zeiss, Oberkochen, Germany) equipped with ZEN Lite imaging software.

Shwab E.K., Juvvadi P.R., Waitt G., Shaheen S., Allen J I.V., Soderblom E.J., Bobay B.G., Asfaw Y.G., Moseley M.A, & Steinbach W.J. (2020). The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets. mBio, 11(6), e02880-20.

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Visualizing MTCH2 Mitochondrial Localization

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For immunofluorescence assay to observe MTCH2 cellular localization, A172 cells were cultured on coverslips over 48 h. For Mito-Tracker staining, A172 cells were incubated with the Mito-Tracker dye (Invitrogen, 200 nM) in DMEM at 37 °C incubator for 15 min (mins). Then, cells were fixed with 4% paraformaldehyde (PFA, Sigma) solution at room temperature for 30 min. After permeabilization with 0.25% Triton X-100 in PBS, cells were blocked with 10% goat serum and incubated with primary antibodies (anti-MTCH2, Proteintech) overnight at 4 °C. For F-actin staining, 20 nM rhodamine-phalloidin (Thermo) diluted with PBS was applied to PFA-fixed cells that were permeabilized. Then cells were washed with PBS for three times and incubated with ALEXA FLUOR 488/594 secondary antibodies, and mounted slides using ProLong® Gold Antifade Reagent with DAPI (Invitrogen). Images were acquired using Olympus BX63 microscope.

Yuan Q., Yang W., Zhang S., Li T., Zuo M., Zhou X., Li J., Li M., Xia X., Chen M, & Liu Y. (2021). Inhibition of mitochondrial carrier homolog 2 (MTCH2) suppresses tumor invasion and enhances sensitivity to temozolomide in malignant glioma. Molecular Medicine, 27, 7.

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Multiparametric Live Cell Imaging

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Cells (treated or untreated) were incubated with Mitotracker dye (75 nM, Invitrogen), Lysotracker dye (100 nM, Invitrogen) and Hoechst (10 µg/ml) in complete medium and incubated for 30 min at 37 °C in 6 well plate. The cells were then washed with 1X PBS for three times and phenol red free complete media was added for imaging under live cell condition. Imaging was done using florescent microscopy (Zeiss Microsystems, GmBH, Germany).

Ahmad I., Pal S., Singh R., Ahmad K., Dey N., Srivastava A., Ahmad R., Suliman M., Alshahrani M.Y., Barkat M.A, & Siddiqui S. (2023). Antimicrobial peptide moricin induces ROS mediated caspase-dependent apoptosis in human triple-negative breast cancer via suppression of notch pathway. Cancer Cell International, 23, 121.

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Mitochondrial Membrane Potential Assessment

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Cells in 6 cm plates were harvested and washed three times with PBS, followed by resuspension in pre-warmed PBS containing 200 nM Mitotracker dye (Invitrogen, Waltham, MA, USA). After incubation for 30 min at 37 °C, the fluorescence at Ex./Em. = 579/599 nm was detected by flow cytometry.

Zhang H., Chen Y., Liu X, & Deng H. (2023). Multi-Omics Analyses Reveal Mitochondrial Dysfunction Contributing to Temozolomide Resistance in Glioblastoma Cells. Biomolecules, 13(9), 1408.

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